Summary Accurate control of genome replication initation is essential for genome stability. In particular, organisms with multiple replication origins per chromosome must ensure that initiation of DNA replication at each origin is highly regulated. In eukaryotes, the cell cycle circuitry controls initiation by a complex series of post- translation modifications. The manner in which these modifications impinge upon the function of the replication apparatus is an area of active research and much remains to be discovered. We have previously shown that archaea of the genus Sulfolobus possess three replication origins per chromosomes and each fires once in every cell cycle. In the current proposal we describe a series of experiments to investigate the control of loading and activation of the conserved MCM replication helicase. In eukaryotic chromosomes, all replication start sites are defined by binding of the origin recognition complex, ORC that ultimately directs the position of loading of the MCM helicase. Given their central role in replication it is not surprising that components of ORC, such as Orc1, are essential genes in eukaryotes. Orc1 is evolutionarily conserved in the archaeal domain of life. The chromosomes of archaea of the genus Sulfolobus contain three replication origins that are defined by distinct initiator proteins. Furthermore none of the Sulfolobus Orc1-like initiators are essential for viability. This mosaic nature of the Sulfolobus chromosome provides a unique tool with which to dissect the mode of action of the conserved Orc1 proteins in living cells. We will exploit this tool to perform genetic biochemical and dissections of the role of Orc in MCM loading in vivo and in vitro. Additionally, our preliminary data reveal the existence of positive control of the MCM helicase by phosphorylation and we will determine the basis of this activation of the helicase. In addition to establishing novel paradigms for prokaryotic DNA replication, our work will serve as a model for the fundamentally related, yet organizationally more complex, eukaryotic DNA replication apparatus.